Sample processing devices, and methods of use thereof

ABSTRACT

Embodiments of the invention provide a filter kit including filters for processing a biological sample. Some embodiments include a filter cap in a tube kit with a first tube containing a buffer solution and a second tube containing a lyophilized master mix. Some embodiments include a method of processing a sample using the kit including mixing a biological sample in a first tube with the buffer solution, positioning the filter cap in the first tube, positioning a second tube on the filter cap, flipping the first tube, the filter cap, and the second cap to filter the biological sample and buffer solution mixture with the filter cap as it flows from the first tube to the second tube. Some embodiments include structure enabling transfer of materials through inline flow between the tubes. Some further embodiments include integrated structure for sample pulverization with integrated buffer and lyophilized master mix.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/579,887, filed Dec. 5, 2017, which is a 371 National Stageapplication of PCT/US2016/036050, filed Jun. 6, 2016, which claims thebenefit of and priority to U.S. Provisional Application No. 62/171,355,filed on Jun. 5, 2015, the entire contents of which are incorporatedherein by reference.

BACKGROUND

The processing of raw biological samples for extraction of DNA forgenetic profiling usually requires multiple vessels that are usedthrough a plurality of processing steps. Typically, separate vessels areused for one or more different biochemical analysis steps, includingtaking the sample, disrupting the sample with chemical or mechanicalmeans, extracting DNA from the sample, and analyzing the sample. Notonly is the use of multiple vessels time-consuming and costly, theintroduction of multiple surfaces and transfer of samples from onevessel to another can increase the risk of contamination that canintroduce errors during the downstream sample analysis step.Accordingly, there is a need to integrate and miniaturize various sampleprocessing steps into a unitary or modular device, to significantlyreduce or eliminate the need to transfer biological samples betweenmultiple separate vessels. A modular design can increase the efficiencyof extracting nucleic acids from tissue samples, and increase efficiencyby largely eliminating the need to transfer reactants between separatereaction vessels to perform each step of the extraction process.Furthermore, integration of the device with external automationequipment modular designs can allow the extraction process to beperformed with only one action from the user, thereby enabling theprocess to be fully automated after the user initiates the extractionprocess.

SUMMARY

Some embodiments include a filter kit comprising a center portion, and afirst connection portion coupled to a first side of the center portion,where the first connection portion is coupled to a first tube containinga buffer solution. Further, a second connection portion is coupled to asecond side of the center portion, where the second connection portionis coupled to a second tube containing a lyophilized master mix.Further, the filter cap includes a first filter at least partiallylocated in the first connection portion, and a second filter at leastpartially located in the second connection portion. In some furtherembodiments, the filter cap comprises a third filter at least partiallylocated in the center portion of the filter cap.

Some embodiments include a tube kit comprising a first tube containing abuffer solution, and a second tube containing a lyophilized master mix.The kit also includes a filter cap comprising a center portion, a firstconnection portion coupled to a first side of the center portion, and asecond connection portion coupled to a second side of the centerportion. Further, the kit also includes a first filter at leastpartially located in the first connection portion, and a second filterat least partially located in the second connection portion.

In some embodiments, the first connection portion of the filter cap isconfigured to be positioned in a cavity in the first tube to form atleast a partial mechanical seal with the first tube. In some furtherembodiments, the second connection portion of the filter cap isconfigured to be positioned in a cavity in the second tube to form atleast a partial mechanical seal with the second tube.

Some embodiments include a first adhesive on the first side of thecenter portion of the filter cap around the first connection portion,where the first adhesive is configured to form a seal between the filtercap and the first tube. Further, a second adhesive on the second side ofthe center portion of the filter cap around the second connectionportion is configured to form a seal between the filter cap and thesecond tube.

Some embodiments include a method comprising placing a biological samplein a first tube that is preloaded with a buffer solution, where abiological sample and buffer solution mixture is formed. Further, themethod includes positioning a filter cap in the first tube, positioninga second tube on the filter cap, flipping the first tube, the filtercap, and the second cap, and filtering the biological sample and buffersolution mixture with the filter cap as it flows from the first tube tothe second tube. Some further embodiments include positioning the filtercap in the first tube that includes positioning a first connectionportion of the filter cap in the first tube. Some other embodimentsinclude positioning a second tube on the filter cap that includespositioning a second tube on a second connection portion of the filtercap.

Some embodiments of the invention include a tube kit comprising a firsttube containing a buffer solution, where there is an opening at a firstend of the first tube and a seal across a second end of the first tube.Further, the tube kit comprises a second tube containing a lyophilizedmaster mix, and a lid with a tip coupled to the lid, where the secondend of the first tube is configured to form a seal with the first end ofthe second tube, and where the tip coupled to the lid is configured topuncture the seal on the second end of the first tube as the lid isplaced on the first tube.

In some embodiments, the first tube sits in the second tube. In somefurther embodiments, the first tube clips onto the second tube. In otherembodiments, the lid has a bulb top. In some further embodiments, thesecond tube includes a filter.

Some embodiments include a method comprising adding a biological sampleto a first tube containing a buffer solution, where the biologicalsample is added through an opening at a first end of the first tube, andpositioning a second end of the first tube in a second tube, puncturinga seal on the second end of the first tube with a tip coupled to a lid,and positioning the lid on the first tube to form a seal between the lidand the first tube.

Some embodiments include an inline sample processing device comprising afirst chamber configured to receive a tissue sample and a nucleic acidextraction reagent, where the first chamber is configured to permit thetissue sample to be pulverized. Further, the method includes a secondchamber in flow communication with the first chamber that is configuredto receive the extraction reagent and a portion of the pulverized tissuesample. Further, the device includes a first separation element disposedbetween the first chamber and the second chamber and configured topermit a portion of the pulverized tissue sample and the extractionreagent to flow from the first chamber to the second chamber. Further, athird chamber is in flow communication with the second chamber, and hasa plurality of ports configured to allow a reagent to be delivered tothe third chamber, and a second separation element is disposed betweenthe second chamber and the third chamber and is configured tosubstantially prevent the pulverized tissue from entering the thirdchamber.

Some embodiments include a sample processing device for pulverizationand denaturation comprising a hollow cylindrical portion, a hollowtapered portion, a first cap for covering an end of the cylindricalportion, and a second cap for covering an end of the tapered portion.Further, the device can include a plurality of ribs extending along thecylindrical portion, a ring at an end of the plurality of ribs where thecylindrical portion meets the tapered portion, and a pulverizercontained within the cylindrical portion between the ring and the firstcap.

In some embodiments, the pulverizer is cylindrical or spherical. In somefurther embodiments, the pulverizer is glass, ceramic, stainless steel,or a non-reactive polymer. In some other embodiments, the plurality ofribs prevents the pulverizer from touching sides of the sampleprocessing device and allows the pulverizer to slide within thecylindrical portion. In some further embodiments, the ring prevents thepulverizer from entering the tapered portion of the sample processingdevice.

Some embodiments include a method for pulverizing and denaturing a crudesample comprising placing a pulverizer in a sample processing device.The device can comprise a cylindrical portion, a tapered portion, and aplurality of ribs extending along the cylindrical portion, and a ring atan end of the plurality of ribs where the cylindrical portion meets thetapered portion. The method can include placing the crude sample in thecylindrical portion of the sample processing device, sealing the sampleprocessing device, and shaking the sample processing device such thatthe pulverizer macerates the crude sample to create a pulverized sample.Further, the method can include adding a denaturing agent into thetapered portion of the sample processing device, sealing the sampleprocessing device, and shaking the sample processing device such thatthe denaturing agent denatures DNA within the pulverized sample tocreate a denatured sample. Some embodiments comprise transferring thedenatured sample into a testing vessel. Other embodiments includetesting the denatured sample in the sample processing device.

Some embodiments of the invention include the sample processing devicecomprising a tube comprising a removable basket with a grid and aplurality of tabs that hang over an edge of the tube, a barcode, and acap comprising a ring-shaped punch edge for punching a sample out of acrude biological material, and a plurality of fingers or teeth forforcing the sample through the grid of the removable basket in the tube.

In some embodiments of the invention, the cap snaps onto the tube. Inother embodiments, the cap twists onto the tube. Some furtherembodiments include a sample processing liquid. In other embodiments,the sample processing liquid is a lysis buffer in the tube. In somefurther embodiments, the sample processing liquid is sodium hydroxidecontained within a pouch in the tube or the cap. In other embodiments,the tube further comprises a permeable layer adjacent to the sodiumhydroxide pouch. In some embodiments, the cap further comprises apiercer. In some further embodiments, the cap further comprises aseptum.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is side view of a first tube in accordance with some embodimentsof the invention.

FIG. 1B is a side view of a second tube in accordance with someembodiments of the invention.

FIG. 1C is a side view of a filter cap in accordance with someembodiments of the invention.

FIG. 2 is a flow chart showing the steps of preparing a biologicalsample for testing in accordance with some embodiments of the invention.

FIG. 3 is a side view of the filter cap positioned on the first tube inaccordance with some embodiments of the invention.

FIG. 4 is a side view of the filter cap positioned between the firsttube and the second tube in accordance with some embodiments of theinvention.

FIG. 5 is a side view of the filter cap positioned between the firsttube and the second tube in accordance with some embodiments of theinvention.

FIG. 6A is side view of a first tube in accordance with some furtherembodiments of the invention.

FIG. 6B is a side view of a second tube in accordance with some furtherembodiments of the invention.

FIG. 6C is a side view of a lid in accordance with some furtherembodiments of the invention.

FIG. 7 is a flow chart showing the steps of preparing a biologicalsample for testing in accordance with some embodiments of the invention.

FIG. 8 is a side view of the first embodiment of the first tubepositioned in the first embodiment of the second tube in accordance withsome embodiments of the invention.

FIG. 9 is a side view of the first embodiment of the first tubepositioned in the first embodiment of the second tube with the firstembodiment of the lid being placed in the first tube in accordance withsome embodiments of the invention.

FIG. 10 is a side view of the first embodiment of the first tubepositioned in the first embodiment of the second tube with the firstembodiment of the lid fully placed on the first tube in accordance withsome embodiments of the invention.

FIG. 11A is a side view of a second embodiment of a lid.

FIG. 11B is a side view of the first embodiment of the first tubepositioned in the first embodiment of the second tube with the secondembodiment of the lid being placed in the first tube in accordance withsome embodiments of the invention.

FIG. 12A is a side view of a second embodiment of the second tube.

FIG. 12B is a side view of the first embodiment of the first tubepositioned in the second embodiment of the second tube with the firstembodiment of the lid being placed in the first tube in accordance withsome embodiments of the invention.

FIG. 13A is a cross-sectional view of a first tube in accordance withsome further embodiments of the invention.

FIG. 13B is a cross-sectional view of a second tube in accordance withsome other embodiments of the invention.

FIG. 13C is a cross-sectional view of the second embodiment of the firsttube positioned in the third embodiment of the second tube in accordancewith some embodiments of the invention.

FIG. 13D is a cross-sectional view of the second embodiment of the firsttube positioned in the third embodiment of the second tube with thefirst embodiment of the lid being placed in the first tube in accordancewith some embodiments of the invention.

FIG. 14 is a plan view of a multi-chamber inline sample processingdevice in accordance with some embodiments of the invention.

FIG. 15A is a plan view of the sample processing device showing a tissuesample inserted in the pulverization chamber in accordance with someembodiments of the invention.

FIG. 15B is a plan view of the sample processing device showing thepulverization chamber with a cap coupled and shows the tissue sample ina pulverized state in accordance with some embodiments of the invention.

FIG. 16 is a plan view of the sample processing device showing anextraction chamber with the tissue sample and an extraction bufferdisposed therein in accordance with some embodiments of the invention.

FIG. 17A is a plan view of the sample processing device showing adilution chamber in accordance with some embodiments of the invention.

FIG. 17B is a plan view of the sample processing device showing adiluted isolated nucleic acid sample in accordance with some embodimentsof the invention.

FIG. 18 is a flow diagram showing a method for performing an inlineextraction of a nucleic acid from a tissue sample in accordance withsome embodiments of the invention.

FIG. 19 is side view of a sample processing device for pulverizing anddenaturing in accordance with some embodiments of the invention.

FIG. 20 is a cross-sectional view of the sample processing device ofFIG. 19 along line A-A.

FIG. 21A is a cross-sectional view of one embodiment of the sampleprocessing device of FIG. 19.

FIG. 21B is a cross-sectional view of another embodiment of the sampleprocessing device of FIG. 19.

FIG. 22 is a perspective view of a sample processing device inaccordance with some embodiments of the invention.

FIG. 23 is a perspective view of another embodiment of the sampleprocessing device of FIG. 22.

FIG. 24 is a perspective view of another embodiment of the sampleprocessing device of FIG. 22.

FIG. 25 is a perspective view of another embodiment of the sampleprocessing device of FIG. 22.

FIG. 26 is a perspective view of another embodiment of the sampleprocessing device of FIG. 22.

FIG. 27 is a perspective view of another embodiment of the sampleprocessing device of FIG. 22.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the following drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways. Also, it is to be understood thatthe phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having” and variations thereof herein ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items. Unless specified or limited otherwise, theterms “mounted,” “connected,” “supported,” and “coupled” and variationsthereof are used broadly and encompass both direct and indirectmountings, connections, supports, and couplings. Further, “connected”and “coupled” are not restricted to physical or mechanical connectionsor couplings. Unless specified or limited otherwise, the terms “tube” or“tubes, and variations thereof are used broadly and encompass anycross-sectional shape and/or any shape of container.

The following discussion is presented to enable a person skilled in theart to make and use embodiments of the invention. Various modificationsto the illustrated embodiments will be readily apparent to those skilledin the art, and the generic principles herein can be applied to otherembodiments and applications without departing from embodiments of theinvention. Thus, embodiments of the invention are not intended to belimited to embodiments shown, but are to be accorded the widest scopeconsistent with the principles and features disclosed herein. Thefollowing detailed description is to be read with reference to thefigures, in which like elements in different figures have like referencenumerals. The figures, which are not necessarily to scale, depictselected embodiments and are not intended to limit the scope ofembodiments of the invention. Skilled artisans will recognize theexamples provided herein have many useful alternatives that fall withinthe scope of embodiments of the invention.

FIG. 1A is side view of first tube 100 in accordance with someembodiments of the invention. In some embodiments, the first tube 100includes body 102 at least partially enclosing or forming the cavity104, and a lid 106. As illustrated in FIG. 1A, in some embodiments, thelid 106 can be coupled or tethered to the body 102. In some otherembodiments, the lid 106 can be detached from or independent from thebody 102, and later coupled to the body 102 when required. For example,in some embodiments, the lid 106 can be sized to be positioned in thecavity 104 to at least partially seal the first tube 100. In someembodiments, the lid 106 can be coupled to the body 102 using anyconventional coupling. FIG. 1B is a side view of second tube 110 inaccordance with some embodiments of the invention. In some embodiments,the second tube 110 shown in FIG. 1B can include a body 112, cavity 114,and a lid 116. In some embodiments, the lid 116 can be coupled to thebody 112. In some other embodiments, the lid 116 can be detached fromthe body 112, and later coupled to the body 112 when required. Forexample, in some embodiments, the lid 116 can be sized to be positionedin the cavity 114 to at least partially seal the first tube 110. In someembodiments, the lid 116 can be coupled to the body 112 using anyconventional coupling. In some further embodiments of the invention,body 102 and/or body 112 can be used without a lid (i.e., lid 106 and/orlid 116 can be absent). In some embodiments, the first tube 100 and/orthe second tube 110 and include a generally rounded or curved end. Inother embodiments, the first tube 100 and/or the second tube 110 cancomprise a closed end that comprises a flat or square end.

Referring again to FIG. 1A, in some embodiments, the first tube 100 cancontain a volume of buffer solution B in cavity 104 of the body 102. Insome embodiments, the volume of buffer solution B can at least partiallyfill the first tube 100 (shown in this non-limiting embodiment asfilling the closed end of the first tube 100 including the rounded orcurved portion). In other embodiments, more or less volume of buffersolution B can be added or included in the first tube 100 (e.g., in akit). Referring again to FIG. 1B, in some embodiments, the second tube110 can contain a lyophilized master mix LMM in cavity 114 of the body112. In some embodiments, the volume of lyophilized master mix LMM canat least partially fill the second tube 110 (shown in this non-limitingembodiment as at least partially filling the closed end of the secondtube 110 including the rounded or curved portion). In other embodiments,more or less volume of lyophilized master mix LMM can be added orincluded in the second tube 110.

FIG. 1C is a side view of filter cap 120 in accordance with someembodiments of the invention. In some embodiments, the filter cap 120can include a first connection portion 122, a second connection portion124, and a center portion 126 positioned substantially between the firstconnection portion 122 and the second connection portion 124. In someembodiments, the filter cap 120 can be coupled with the first tube 100and/or the second tube 110 and used to perform one or more filteringfunctions of material within the tubes 100, 110. In some embodiments,the first connection portion 122 can be coupled to a first side ofcenter portion 126 and second connection portion 124 is coupled to asecond side of center portion 126. Further, in some embodiments, thefirst connection portion 122, and/or the second connection portion 124,and/or the center portion 126 can include one or more filters. In someembodiments, the first connection portion 122 of filter cap 120 can besized to be positioned in cavity 104 of the first tube 100 to form atleast a partial seal between the filter cap 120 and the first tube 100.Further, in some embodiments, the second connection portion 124 offilter cap 120 can be sized to be positioned in cavity 114 of the secondtube 110 to form at least a partial seal between filter cap 120 andsecond tube 110. In some embodiments of the invention, the filter cap120 can include a first adhesive 128 on the first side of center portion126 around first connection portion 122, and a second adhesive 129 onthe second side of center portion 126 around the second connectionportion 124. In some embodiments, the first adhesive 128 and secondadhesive 129 can hold the filter cap 120 in position in the first tube100 and the second tube 110 when the filter cap 120 is positioned in thefirst tube 100 and second tube 110. Further, in some embodiments, thepositioning of the filter cap 120 with adhesives 128, 129 can preventleakage between the filter cap 120 and the first tube 100 and secondtube 110. In an alternate embodiment of the invention, the firstadhesive 128 and/or second adhesive 129 can be omitted, and the filtercap 120 can at least partially seal against first tube 100 and secondtube 110 using a conventional mechanical seal.

FIG. 2 is a flow chart showing the steps of preparing biological sampleS for testing in accordance with some embodiments of the invention. Insome embodiments, FIG. 2 includes steps 130, 132, 134, 136, and 138. Insome embodiments, the steps of preparing biological sample S for testingcan include the order as shown. In other embodiments, any of the steps130, 132, 134, 136, 138 can proceed in a different order than shown.Further, in some embodiments, any of the steps 130, 132, 134, 136, 138can be included, omitted, or repeated.

In some embodiments of the invention, the filter cap 120 can bepositioned between the first tube 100 and the second tube 110 to enablefiltering a biological sample. For example, FIGS. 3-5 show steps 132,134, and 138, respectively. FIG. 3 is a side view of filter cap 120positioned on first tube 100 in accordance with some embodiments of theinvention. FIG. 4 is a side view of filter cap 120 positioned betweenfirst tube 100 and second tube 110 in accordance with some embodimentsof the invention. In some embodiments of the invention, step 130 caninclude placing biological sample S in a first tube 100 that containsbuffer solution B. In some embodiments, first tube 100 can have buffersolution B placed or pre-loaded in first tube 100 prior to thecollection of a biological sample S. In some embodiments, the buffersolution B in the first tube 100 can vary depending on what biologicalsample S is to be tested. In some embodiments of the invention, thebiological sample S can be pipetted into the first tube 100 to mix withbuffer solution B in the first tube 100. In some embodiments, this cancreate a biological sample S and buffer solution B mixture (S+B). Insome embodiments, the biological sample and buffer solution mixture S+Bcan be heated in the first tube 100 if needed.

In some embodiments of the invention, with the lid 106 positioned awayfrom the open end of the body 102, step 132 can include positioning afirst connection portion 122 of filter cap 120 in first tube 100, asseen in FIG. 3. In some embodiments, the first connection portion 122 offilter cap 120 can form at least a partial seal with first tube 100. Insome embodiments, first adhesive 128 can be applied to the first side ofcenter portion 126 of filter cap 120 around the first connection portion122 to strengthen the seal between filter cap 120 and first tube 100.Further, in some embodiments, the first adhesive 128 can help to preventbiological sample and buffer solution mixture S+B from leaking out offirst tube 100.

In some embodiments, step 134 can include positioning the second tube110 on the second connection portion 124 of filter cap 120, as seen inFIG. 4. In some embodiments, the second tube 110 can have lyophilizedmaster mix LMM placed or pre-loaded in the second tube 110 prior to thecollection of biological sample S. In some embodiments, lyophilizedmaster mix LMM preloaded in second tube 110 can vary depending on whatbiological sample S is to be tested. In some embodiments, the secondconnection portion 124 of filter cap 120 can form at least a partialseal with the second tube 110. In some embodiments, the second adhesive129 can be applied to the second side of center portion 126 of filtercap 120 around second connection portion 124 to strengthen the sealbetween filter cap 120 and second tube 110.

In some embodiments, step 138 can include filtering the biologicalsample and buffer solution mixture S+B through filter cap 120. In someembodiments of the invention, step 136 can include rotating, turningand/or flipping the first tube 100, filter cap 120, and second tube 110.In some embodiments, the rotating, turning and/or flipping the firsttube 100, filter cap 120, and second tube 110 can include a singlerotation, turn or flip. In other embodiments, the rotating, turningand/or flipping the first tube 100, filter cap 120, and second tube 110can include multiple or repeated rotating, turning and/or flipping. Insome embodiments, the action of rotating, turning and/or flipping canagitate the contents of the first tube 100 and/or the second tube 110.

In some embodiments, this movement can allow the biological sample andbuffer solution mixture S+B to flow through the filter cap 120 into thesecond tube 110. In some embodiments, the second adhesive 129 that ispositioned between filter cap 120 and second tube 110 can help preventbiological sample and buffer solution mixture S+B from leaking out offilter cap 120 and second tube 100 as the biological sample and buffersolution mixture S+B flows through the filter cap 120 into the secondtube 110. For example, FIG. 5 is a side view of filter cap 120positioned between first tube 100 and second tube 110 in accordance withsome embodiments of the invention. In this non-limiting example, thepositions of the first tube 100 and second tube 110 are flipped about180° than that illustrated in FIG. 4, and the biological sample andbuffer solution mixture S+B has exited the first tube 100 and enteredthe second tube 110 through filter cap 120. In some embodiments, thefilter cap 120 can include filters to filter contamination or otherunwanted or undesirable material in the biological sample and buffersolution mixture S+B. In some embodiments, the biological sample andbuffer solution mixture S+B can be filtered through filter cap 120, andcan mix with the lyophilized master mix LMM in the second tube 110. Insome embodiments, this can create a final mixture FM (which includesbiological sample S, buffer solution B, and lyophilized master mix LMM)for testing. In some embodiments of the invention, the filter cap 120can be removed from second tube 110 and lid 116 of second tube 110 canbe positioned in the cavity 114 of the second tube 110 to at leastpartially seal the final mixture FM in the second tube 110. In someembodiments, the second tube 110 can then be positioned in a device fortesting.

In some embodiments, the filter cap 120 can allow fluid to flow in bothdirections. In a further embodiment, the filter cap 120 can act as acheck valve, and can allow fluid to flow single direction (e.g., fromthe first tube 100 to the second tube 110 but not from the second tube110 to the first tube 100). In the method described above for instance,filter cap 120 can allow biological sample and buffer solution mixtureS+B to flow from the first tube 100 to a second tube 110, but not fromthe second tube 110 to the first tube 100.

In some embodiments. the first tube 100, second tube 110, and filter cap120 can allow a user to prepare a crude biological sample S for testingin the field. In some embodiments of the invention, the first tube 100,second tube 110, and filter cap 120 are advantageous, as they can allowa user to prepare biological sample S for testing in the field usingminimal instruments. For example, in some embodiments, the first tube100 and the second tube 110 can be conventional or standard tubes thatare preloaded with a buffer solution B and lyophilized master mix LMMnecessary for performing a particular task. In some embodiments, thecrude biological sample S can then be placed in the first tube 100 inthe field, and mixed with buffer solution B. In some embodiments, thefilter cap 120 can be positioned on the first tube 100 and second tube110 can be placed on the filter cap 120. In some embodiments, the firsttube 100, second tube 110, and filter cap 120 can then be flipped andagitated so that at least some portion of the biological sample andbuffer solution mixture S+B can flows from the first tube 110 to secondtube 120. In some embodiments, the rotating, turning and/or flipping thefirst tube 100, filter cap 120, and second tube 110 can include at leastone rotation, turn or flip. In some embodiments, the action of rotating,turning and/or flipping can agitate the contents of the first tube 100and/or the second tube 110.

In some embodiments of the invention, the filter cap 120 can filtercontamination or other unwanted or undesirable material out ofbiological sample and buffer solution mixture S+B. In some embodiments,the biological sample and buffer solution mixture S+B that flows intosecond tube 120 can then be mixed with lyophilized master mix LMM insecond tube 120. In some embodiments, this further processes biologicalsample S to prepare biological sample S for testing. Further, in someembodiments, the second tube 110 can be removed from the filter cap 120and lid 116 of second tube 110 can closed. In some embodiments, thesecond tube 110 can then be placed in a device for testing.

FIG. 6A is side view of first tube 200 in accordance with some furtherembodiments of the invention. In some embodiments, the first tube 200can include a body 202 at least partially enclosing a cavity 204.Further, the first tube 200 can include an opening 206 at one end, andseal 208 at an opposite end. In some embodiments, the first tube 200 cancomprise a generally square or flat end proximate the seal 208. FIG. 6Bis a side view of second tube 210 in accordance with some furtherembodiments of the invention. In some embodiments, the second tube 210can include a body 212, cavity 214 formed in the body 212, and anopening 216 positioned at a first end of the cavity 214. In thenon-limiting example embodiments shown, the second tube 210 can includea closed end comprising a generally rounded or curved end. In someembodiments, the first tube 200 can contain a buffer solution B in thecavity 204 of body 202. Further, in some embodiments, the second tube210 can contain a lyophilized master mix LMM positioned in the cavity214 of body 212.

FIG. 6C is a side view of lid 220 in accordance with some furtherembodiments of the invention. In some embodiments, the lid 220 caninclude a lid portion 222 and pipette tip 224. In some embodiments, thepipette tip 224 is coupled to and extends from a first side of lidportion 222 of the lid 220. In some embodiments of the invention, thelid 220 can be positioned in a tube (e.g., such as tubes 200, 210) sothat pipette tip 224 can extend into the tube, and the lid portion 222forms at least a partial seal with at least a portion of the tube.

In some embodiments of the invention, the first tube 200 and/or thesecond tube 210 and/or the pipette tip 224 can be configured to processa sample (e.g., such as a biological sample). For example, FIG. 7 is aflow chart showing the steps of preparing biological sample S fortesting in accordance with some embodiments of the invention. FIG. 7includes steps 230, 232, 234, 236, and 238. In some embodiments, thesteps of preparing biological sample S for testing can include the orderas shown. In other embodiments of the invention, any of the steps 230,232, 234, 236, 238 can proceed in a different order than shown. Further,in some embodiments, any of the steps 230, 232, 234, 236, 238 can beincluded, omitted, or repeated.

FIGS. 8-10 illustrate views representing at least one or more of theprocess steps of FIG. 7. FIG. 8 is a side view of the first tube 200positioned in the second tube 210 in accordance with some embodiments ofthe invention, and FIG. 9 is a side view of the first tube 200positioned in the second tube 210 with lid 220 being placed in the firsttube 200 in accordance with some embodiments of the invention. Further,FIG. 10 is a side view of the first tube 200 positioned in second tube210 with lid 220 fully placed on the first tube 200 in accordance withsome embodiments of the invention.

Referring to FIG. 7, step 230 can include adding biological sample S toa first tube 200 that contains buffer solution B. As illustrated in FIG.6A, buffer solution B can be placed in the first tube 200 prior to thecollection of biological sample S. In some embodiments, the buffersolution B in first tube 200 can vary depending on the type ofbiological sample S. In some embodiments, the biological sample S can bepipetted into opening 206 of first tube 200 to mix with the buffersolution B in the cavity 204 to create a biological sample S and buffersolution B mixture (B+S).

In some embodiments, step 232 includes positioning a second end of thefirst tube 200 in the second tube 210, as seen in FIG. 8. The second endof first tube 200 has smaller diameter than the diameter of the firstend of second tube 210, thereby allowing the insertion of at least aportion of the first tube 200 into the cavity 214. In some embodiments,the body 202 of first tube 200 include walls that slope based on asmaller diameter of the body 202 at the second end to a larger diameterof the body 202 at the first end. Thus, in some embodiments, when thesecond end of first tube 200 is positioned in the second tube 210 asdescribed earlier, the first tube 200 can at least partially slide intothe second tube 210 until the an outer diameter of first tube 200 isabout the diameter of the inner diameter of the first end of the secondtube 210. In some embodiments, this can form at least a partial sealbetween the first tube 200 and the second tube 210. In some embodimentsof the invention, the second tube 210 contains lyophilized master mixLMM. In some embodiments, the lyophilized master mix LMM in second tube110 can vary depending on what biological sample S is to be tested.

In some embodiments of the invention, step 234 can include positioningthe pipette tip 224 of lid 220 in the first tube 200, as seen in FIG. 9.In some further embodiments of the invention, step 236 can includepuncturing the seal 208 on the first tube 200 with the pipette tip 224of lid 220. In some embodiments, the pipette tip 224 of lid 220 can belonger than the first tube 200, and thus in some embodiments, as thepipette tip 224 of lid 220 is positioned in first tube 200, the pipettetip 224 can contact the seal 208 of the first tube 200. In someembodiments of the invention, the pipette tip 224 can puncture the seal208 of the first tube 200 as pipette tip 224 is moved through the firsttube 200 and into the second cavity 214. In an alternative embodiment,pipette tip 224 can be a sharp tip capable of puncturing seal 208 butwithout pipetting capabilities. In some embodiments, the puncturing seal208 can cause biological sample and buffer solution B+S to flow fromfirst tube 200 into second tube 210. In some embodiments, the biologicalsample and buffer solution B+S can then mix with lyophilized master mixLMM to form a final mixture FM that is suitable for testing.

In some embodiments, step 238 can include positioning the lid portion222 of lid 220 on the first tube 200 to form at least a partial sealbetween the lid 220 and the first tube 200, as seen in FIG. 10. Further,as the pipette tip 224 is moved as described above, after the pipettetip 224 punctures the seal 208, it can extend through the first tube 200and into the second tube 210 until the lid portion 222 of lid 220 comesinto contact with first tube 200. In some embodiments, this can form aseal between the lid 220 and the first tube 200. Further, in someembodiments, the lid 220, first tube 200, and second tube 210 can thenbe placed in a device for testing.

In some embodiments of the invention, the first tube 200, second tube210, and lid 220 can allow a user to prepare a crude biological sample Sfor testing in the field. In some embodiments, the lid 220, first tube200, and second tube 210 are advantageous, as they allow a user toprepare biological sample S for testing in the field using minimalinstruments. For example, in some embodiments, the first tube 200 andthe second tube 210 can have a buffer solution B and lyophilized mastermix LMM added to them, and can then be taken into the field. In someembodiments, a crude biological sample S can then be collected in thefield and placed in the first tube 200, and mixed with buffer solutionB. In some embodiments of the invention, the first tube 200 can then bepositioned in second tube 210 to form at least a partial seal betweenfirst tube 200 and second tube 210. In some embodiments, the pipette tip224 of lid 220 can then be positioned in first tube 200 and movedthrough first tube 200. In some embodiments, as the pipette tip 224moves through first tube 200, it can contact with and puncture the seal208 on first tube 200 as described earlier. In some embodiments, thiscan cause the field-sampled biological sample and buffer solutionmixture B+S to flow into the second tube 210, where the biologicalsample and buffer solution mixture B+S can mix with the lyophilizedmaster mix LMM. In some embodiments, the steps as described can furtherprocess the biological sample S for testing by placing the Lid 220,first tube 200, and second tube 210 in a testing device.

In some embodiments, the first tube 200 and the second tube 210 can beused with alternative types of lid structures. For example, FIG. 11A isa side view of the lid 220A in accordance with some embodiments of theinvention, and FIG. 11B is a side view of the first tube 200 positionedin the second tube 210 with lid 220A being placed in the first tube 200in accordance with some embodiments of the invention. In someembodiments, the lid 220A can include a lid portion 222A, pipette tip224A, and bulb portion 226A. In some embodiments, the pipette tip 224Acan be coupled to the first side of the lid portion 222A, and the bulbportion 226A can be coupled to a second side of the lid portion 222A. Insome embodiments, the bulb portion 226A can include a cavity that iscoupled to a cavity of pipette tip 224A. In some embodiments, thisstructure can enable the lid 220A to act as a pipette. For example, insome embodiments, a user can grasp and squeeze the bulb portion 226A,place the pipette tip 224A in a fluid, and release the bulb portion 226Ato suck the fluid into the pipette tip 224A. In some embodiments, theuse of the lid 220A with the first tube 200 and the second tube 210 canallow a user to use the lid 220A to pipette the biological sample S intothe first tube 200 to mix with buffer solution B. In some embodiments,the pipette tip 224A of lid 220A can then be used to puncture the seal208 in the first tube 200 and lid portion 222A of lid 220A can sealagainst first tube 200, as described in reference to FIGS. 6A-10 above.

In some embodiments, the second tube 210 can include an integratedfilter. For example, FIG. 12A is a side view of a second tube 210A inaccordance with some embodiments of the invention. FIG. 12B is a sideview of the first tube 200 positioned in the second tube 210A with thelid 220 being placed in the first tube 200 in accordance with someembodiments of the invention. In some embodiments, the second tube 210Acan include body 212A, cavity 214A formed by the body 212A, opening216A, and filter 218A positioned in the cavity 214A extending from oneside of the body 212A to an opposite side of the body 212A. In someembodiments of the invention, the opening 216A can be positioned at afirst end of the cavity 214A in the second tube 210A. In someembodiments of the invention, the second tube 210A can containlyophilized master mix LMM in the cavity 214A of the body 212A of thesecond tube 210A. In some embodiments, the steps and processes describedabove in FIGS. 6A-10 can be performed with the first tube 200 incombination with a second tube including an integrated filter. Forexample, as seen in FIG. 12B, the first tube 200 can be positioned abovethe filter 218A in the second tube 210A. In some embodiments, once theseal 208 of the first tube 200 is punctured, a biological sample andreagent mixture B+S can flow from the first tube 200 through the filter218A into the second tube 210A. In some embodiments, the filter 218A canfilter contamination or other unwanted or undesirable material in thebiological sample and buffer solution mixture S+B. In some embodiments,filtering contamination or other unwanted or undesirable material fromthe biological sample and reagent mixture B+S can improve the accuracyof testing of biological sample and reagent mixture B+S.

FIG. 13A is a cross-sectional view of a first tube 200A in accordancewith some further embodiments of the invention. In some embodiments, thefirst tube 200A can include body 202A at least partially enclosing thecavity 204A, an opening 206A at one end of the cavity 204A, and a seal208A at an opposite end of the cavity 204A, and flange 209A. In someembodiments, the first tube 200A further contains flange 209A positionedaround an outside diameter of body 202A. In some embodiments, the flange209A can be positioned between the first end and the second end of firsttube 200A. In some embodiments, the flange 209A can include a flat faceon a first side and an angled face on a second side. FIG. 13B is across-sectional view of a second tube 210B in accordance with some otherembodiments of the invention. In some further embodiments, the secondtube 210B can include body 212B, cavity 214B, opening 216B, and flange219B. In some embodiments, the first tube 200A and second tube 210B canbe used to process a sample using a lid such as the lid 220. Forexample, FIG. 13C is a cross-sectional view of the first tube 200Apositioned at least partially in the second tube 210B in accordance withsome embodiments of the invention, and FIG. 13D is a cross-sectionalview of first tube 200A positioned in second tube 210B with the lid 220being placed in first tube 200A in accordance with some embodiments ofthe invention. In some embodiments, the first tube 200A can containbuffer solution B in cavity 204A of body 202A of first tube 200A, andwhen the first tube 200A is positioned in the second tube 210B, as seenin FIGS. 13C-13D, the flanges 209A can engage the flanges 219B toprevent first tube 200A from being pulled out of second tube 210B. Insome embodiments, as the first tube 200A is positioned in the secondtube 210B, the angled faces of flanges 209A can slide against the angledfaces of flange 219B.

In some embodiments, as the first tube 200A moves into the second tube210B, the faces of flanges 209A can slide at least partially past orover the angled faces of flange 219B. In some embodiments, the flanges209A can couple with the inner surface of the second tube 210B, and/orat least a portion of the flange 219B. In other embodiments, anyconventional coupling mechanism can be used to couple the first tube200A to the second tube 210B, and can be used to substantially retainthe first tube 200A in the second tube 210B as shown in FIGS. 13C-13D.

In some embodiments, once the first tube 200A fully positioned in thesecond tube 210B, the flat face of the flange 209A can sit against theflat face of flange 219B. In some embodiments, this engagement canprevent the first tube 200A from being removed from the second tube210B, and can also prevent contamination from passing into second tube210B. In reference to FIG. 13C, in some embodiments, after the firsttube 200A is positioned in the second tube 210B, the pipette tip 224 ofthe lid 220 can then be pushed through first tube 200A to puncture seal208A on first tube 200A, and the lid 220 can at least partially sealagainst the first tube 200A. In other embodiments, the variousembodiments of first tube 200, second tube 210, and lid 220 described inFIGS. 6-13D above can be integrated with one another in any way.

FIG. 14 is a plan view of multi-chamber inline sample processing device300 in accordance with some embodiments of the invention. In someembodiments, the sample processing device 300 can include three inlinechambers, which together define an inline flow path of a materialthrough the sample processing device 300. In some embodiments of theinvention, the sample processing device 300 can include cap 302,pulverization chamber 304, first separation element 306, extractionchamber 308, second separation element 310, and dilution chamber 312. Insome embodiments, the size and volume of each chamber of device 300 canbe scaled to match the quantity of material to be processed. In thisway, each of chambers, 304, 308, and 312 can be sized according to thequantity of material to be processed. For example, in some embodiments,chamber 304 can be sized smaller to process single seeds or leaf punchesor sized larger to accommodate a collection of seeds or other materialthat form a representative sample of a large collection of material tobe analyzed. Furthermore, each of the chambers 304, 308, 312, cap 302,and separation elements 306, 310 of device 300 can be sizedindependently based on the application and quantity of materials needed.

In some embodiments of the invention, the sample processing device 300can be formed as a single piece. In some embodiments, the sampleprocessing device 300 can be formed using a mold and first separationelement 306 and second separation element 310 can be press fit into thesample processing device 300. In other embodiments, each of the chambers304, 308, and 312 can be separate modules that when coupled togetherform the sample processing device 300. Additionally, in someembodiments, each of the chambers 304, 308, and 312 can have othershapes other than the frustoconical shapes depicted in FIG. 14. As anexample, each chamber 304, 308, or 312 can have a cylindrical shape.

In some embodiments, the cap 302 can be coupled to the pulverizationchamber 304 at an upstream end of pulverization chamber 304 with respectto a direction F of flow. Terms such as upstream and downstream as usedin this description refer to relative positions along the direction offlow F. In some embodiments, the first separation element 306 can becoupled both to a downstream end of pulverization chamber 304 and to anupstream end of the extraction chamber 308. In some embodiments, thesecond separation element 310 can be coupled both to a downstream end ofextraction chamber 308 and to an upstream end of dilution chamber 312.In operation, a biological sample, such as a plant tissue, seed, or acombination of material from different sources can be pulverized ormechanically distressed in pulverization chamber 304. In someembodiments, a portion of the pulverized tissue can be passed throughthe first separation element 306 and into the extraction chamber 308. Insome embodiments of the invention, nucleic acids can be extracted fromthe plant tissue within the extraction chamber 308. In some embodiments,the extracted nucleic acids can then pass through the second separationelement 310 and into the dilution chamber 312. In some embodiments, inthe dilution chamber 312, the extracted nucleic acids can be neutralizedwith a neutralization buffer and diluted with water or a dilutionbuffer. The specific structure and operation of each chamber isdescribed in further detail below.

FIG. 15A is a plan view of sample processing device 300 showing tissuesample 314 inserted in pulverization chamber 304 in accordance with someembodiments of the invention. As depicted, in some embodiments, thetissue sample 314 can be any type or mixture of plant tissue, including,but not limited to, seed, leaf, husk or bark, or any other single typeof plant tissue(s) or mixture of plant tissue types. In some embodimentsof the invention, the pulverization chamber 304 can include an upstreamend 316, a downstream end 318, a side wall 320, an upstream opening 322,and a downstream opening 324. In some embodiments, the pulverizationchamber 304 can be defined by an upstream end 316 and a downstream end318, which are joined by side wall 320. In some embodiments of theinvention, the upstream end 316 can define an upstream opening 322,which has a generally circular shape and is sized to permit tissuesample 314 to be inserted into the pulverization chamber 304, wherepulverization of tissue sample 314 can help to break down the componentsof tissue sample 314, such as cell walls, in order to access nucleicacids. As shown, in some embodiments, the pulverization chamber 304 caninclude an overall frustoconical shape, which tapers towards the firstseparation element 306. In some embodiments, the frustoconical shape ofpulverization chamber 304 can help drive materials towards theextraction chamber 308 (not shown in FIG. 15A). In some embodiments, thedownstream end 318 can define a downstream opening 324, which in someembodiments, can permit a flow of materials from pulverization chamber304 to enter the extraction chamber 308.

FIG. 15B is a plan view of the sample processing device 300 showingpulverization chamber 304 with cap 302 coupled and showing the tissuesample 314 in a pulverized state in accordance with some embodiments ofthe invention. As shown in FIG. 15B, in some embodiments, the cap 302can cover an upstream opening 322. In some embodiments, the cap 302 caninclude ports 326, and grinder 328. In some embodiments, the cap 302 canbe a snap-fit or twist-fit on to the upstream end 316.

In operation, a tissue sample 314 can be loaded into the pulverizationchamber 304 and pulverized (e.g., the tissue sample 314 can be broken insmaller pieces.) The tissue sample 314 can be pulverized in differentways. For example, in some embodiments, the side wall 320 ofpulverization chamber 304 can be formed from a flexible material thatcan be compressed by mechanical means (e.g., such as a handheld ormachine operated pliers or other constriction.) As the pulverizationchamber 304 is compressed, the tissue sample 314 can be crushed intosmaller pieces. In some embodiments, to aid in the pulverizationprocess, the interior of the pulverization chamber 304 can be configuredto include an abrasive or corrugated surface, which can help to breakdown tissue sample 314. In some other embodiments, conventional pliers,or other mechanical means can also be inserted into the pulverizationchamber 304 to directly pulverize the tissue sample 314. Additionally,in some further embodiments, an abrasive corrugated surface can beinserted into chamber 304, and a friction between the abrasive surfaceand the grinder 328 of cap 302 can be used to break tissue sample 314into smaller pieces. In some embodiments, the grinder 328 can then bespun to break down tissue sample 314. In some embodiments, the tissuesample 314 can be dehydrated or flash frozen prior to being insertedinto pulverization chamber 304 to make the tissue sample 314 morebrittle and easier to pulverize.

FIG. 16 is a plan view of sample processing device 300 showingextraction chamber 308 with tissue sample 314 and extraction buffer 330disposed therein in accordance with some embodiments of the invention.In some embodiments, the extraction chamber 308 can include an upstreamend 332, a downstream end 334, a side wall 336, an upstream opening 338,and a downstream opening 340, and a first separation element 306. Insome embodiments, the extraction chamber 308 can be defined by theupstream end 332 and the downstream end 334, which are joined by sidewall 336. In some embodiments of the invention, the upstream end 332 cancomprise a frustoconical shape which tapers away from the downstream end334. In some embodiments, the upstream end 332 can define an upstreamopening 338, which permits flow from the pulverization chamber 304 toenter extraction chamber 308. In some embodiments of the invention, thefrustoconical shape of the upstream end 332 can help to dispersecomponents within extraction chamber 308 upon entry. In someembodiments, the downstream end 334 can include a frustoconical shapewhich tapers away from upstream end 332. In some embodiments of theinvention, the downstream end 334 defines a downstream opening 340,which permits flow from extraction chamber 308 to enter dilution chamber312 (not shown). In some embodiments, the frustoconical shape ofdownstream end 334 can help drive components towards dilution chamber312.

In operation, the tissue sample 314 can be pulverized as described abovewith respect to FIGS. 15A and 15B. In some embodiments, once the tissuesample 314 is pulverized, an extraction buffer 330 can be supplied tothe pulverization chamber 330 through one of ports 326, and the otherport 326 can act as a pressure relief vent. In some embodiments of theinvention, the extraction buffer 330 can be any one of many conventionalbuffers or mixtures of conventional buffers, including, but not limitedto, Sodium Hydroxide (NaOH) based buffers. As an example embodiment, asuitable buffer can be formed from NaOH and Sodium Dodecyl (luryl)Sulfate (SDS). SDS is a surfactant that can help to solubilize a cellmembrane, while NaOH can help to break down the cell wall and disruptshydrogen bonding between DNA bases (and thus converting thedouble-stranded DNA into single-stranded DNA.)

In some embodiments of the invention, after a sufficient amount ofextraction buffer 330 is added to pulverization chamber 304, air can besupplied through both of ports 326, shown in order to force materialthough the first separation element 306 and into extraction chamber 308.In other embodiments, a vacuum line can be coupled to one of ports 350shown in FIG. 17A to pull material through first separation element 306instead of using air pressure supplied through ports 326. In somefurther embodiments, air pressure can be supplied through ports 326while a vacuum is applied through ports 350. In another embodiment,centrifuge action can be used to push material through the firstseparation element 306 instead of air pressure or vacuum.

As shown in FIG. 16, in some embodiments of the invention, the firstseparation element 306 can be disposed between the downstream end 318 ofthe pulverization chamber 304 and the upstream end 332 of extractionchamber 308. In some embodiments, the first separation element 306 canbe configured to filter the flow of materials between the pulverizationchamber 304 and extraction chamber 308. As a non-limiting exampleembodiment, the first separation element 306 can be a conventionalfilter or a screen. In some embodiments, the filter or screen can bedesigned to block some of the larger pulverized tissue samples 314 butcan allow individual cells, organelles, etc. to pass through firstseparation element 306. In other embodiments, first separation element306 can be a bursting membrane configured to burst when the pressureinside pulverization chamber 304 (generated by air passed through ports326) exceeds a threshold value. When the membrane bursts, all thecontents of pulverization chamber 304 can be free to enter extractionchamber 308.

In some embodiments, a portion of the pulverized tissue sample 314 andextraction buffer 330 can enter the extraction chamber 308 after passingthrough the first separation element 306. In some embodiments, theextraction buffer 330 can function as stated above to extract nucleicacids such as DNA and/or other components from the pulverized tissuesample 314. In some embodiments of the invention, in order to aid theextraction process, the tissue 314 and extraction buffer 330 inextraction chamber 308 can be heated. The exact temperature to which thetissue 314 and extraction buffer 330 in extraction chamber 308 is heatedcan depend on which extraction buffer 330 is used. Typically, tissue 314and extraction buffer 330 in extraction chamber 308 can be heated toanywhere from about 65 degrees Celsius (about 149 degrees Fahrenheit) toabout 80 degrees Celsius (about 176 degrees Fahrenheit). In someembodiments, heating can be applied either locally to the extractionchamber 308, or generally to the entire device 300. In some embodiments,following the heating, the tissue 314 and the extraction buffer 330 inextraction chamber 308 can be cooled to room temperature. In someembodiments, after the heating and cooling, the extraction chamber 308can include some pulverized tissue sample 314 and extracted nucleicacids 352.

FIG. 17A is a plan view of sample processing device 300 showing dilutionchamber 312. In some embodiments, the dilution chamber 312 can includean upstream end 342, downstream end 344, side wall 346, upstream opening348, and ports 350. FIG. 17A also shows second separation element 310,and extracted nucleic acids 352 and neutralization buffer 354. In someembodiments, the dilution chamber 312 can be defined by the upstream end342 and downstream end 344, which are joined by side wall 346. In someembodiments, the upstream end 342 can include a frustoconical shapewhich tapers away from downstream end 344. In some embodiments, theupstream end 342 defines upstream opening 348, which permits flow fromextraction chamber 308 to enter dilution chamber 312. In someembodiments, the frustoconical shape of upstream end 342 can help todisperse components within dilution chamber 312 upon entry. In someother embodiments of the invention, the downstream end 344 has afrustoconical shape which tapers away from upstream end 342. In someembodiments, the dilution chamber 312 can include ports 350. Asillustrated, in some embodiments, the two of ports 350 can be locatedproximate to upstream end 342, and one of ports 350 can be locatedproximate to the downstream end 344. In some embodiments, thefrustoconical shape of downstream end 344 can help drive componentstowards port 350.

FIG. 17A shows extracted nucleic acid 352 in dilution chamber 312 andpulverized tissue 314 in extraction chamber 308. In order to separateextracted nucleic acid 352 and pulverized tissue 314, in someembodiments, air can be forced through ports 326 causing extractednucleic acid 352 to pass through the second separation element 310. Insome embodiments, the second separation element 310 can be disposedbetween the downstream end 334 of extraction chamber 308 and theupstream end 342 of dilution chamber 312. In some embodiments, thesecond separation element 310 can be a filter or screen that isconfigured to filter smaller particles than first separation element306. Specifically, in some embodiments, the second separation element310 can be sized to not allow pulverized tissue sample 314 to passthrough. Thus, in some embodiments, extracted nucleic acid 352 andpulverized tissue sample 314 can be separated using the process asdescribed.

In other embodiments of the invention, a vacuum line can be coupled toone of ports 350 to pull extracted nucleic acid 352 through secondseparation element 310 instead of using air pressure supplied throughports 326. In some further embodiments, air pressure can be suppliedthrough ports 326 while a vacuum is applied through ports 350. Inanother embodiment of the invention, a centrifuge action can be used topush extracted nucleic acid 352 through second separation element 310instead of air pressure or vacuum.

In some embodiments of the invention, after isolated nucleic acid 352 issupplied to dilution chamber 312, neutralizing buffer 354 can besupplied through one of the ports 350. In some embodiments, theneutralizing buffer 354 can serve to bring the pH of the solution indilution chamber 312 to a suitable level for storing isolated nucleicacid 352. There are many suitable neutralizing buffers that can be usedwith embodiments of the invention, including, but not limited toTris-Hydrochloride (TRIS-HCl).

FIG. 17B is a plan view of sample processing device 300 showing dilutedisolated nucleic acid sample 352. In some embodiments, the solution canbe diluted using water or a buffer. There are many suitable buffers fordilution of nucleic acids. An example of a suitable dilution buffer isTris-EDTA (TE) buffer. In some embodiments, after the isolated nucleicacid 352 is diluted, the dilution chamber 312 can be removed from sampleprocessing device 300 directly, and isolated nucleic acid 352 can bestored therein. In some embodiments, the isolated nucleic acid 352 canalso be dispensed into a vial through port 350. Once in the vial,isolated nucleic acid 352 can be amplified using polymerase chainreaction (PCR) and analyzed using fluorescence scanning techniques. Insome embodiments, isolated nucleic acid 352 can also be aspiratedthrough port 350 directly to a sample vial or an inline amplificationdevice that can amplify the nucleic acids and analyze them usingfluorescence scanning techniques.

FIG. 18 is a flow diagram showing a method 356 for performing an inlineextraction and amplification of a nucleic acid in accordance with someembodiments of the invention. In some embodiments, the method 356 caninclude an insertion step 358, and/or a pulverization step 360, and/oran injection step 362, and/or a filtration step 364, an/or an extractionstep 366, an/or a filtration step 368, and/or a neutralization step 370,and/or a dilution step 372. In other embodiments, any of the steps 358,360, 362, 364, 366, 368, 370, 372 can proceed in a different order thanshown. Further, in some embodiments, any of the steps 358, 360, 362,364, 366, 368, 370, 372 can be included, omitted, or repeated. In someembodiments of the invention, during an insertion step 358, a tissuesample 314 can be loaded into the pulverization chamber 304 of themulti-module inline sample processing device 300. In some embodiments ofthe invention, during a pulverization step 360, the tissue sample 314can be pulverized into a powder. Further, in some embodiments, followingthe step 360, during an injection step 362, an extraction buffer 330 canbe added to the pulverization chamber 304. In some embodiments, thefiltration step 364 can follow the injection step 362. In someembodiments, during the filtration step 364, the solution of extractionbuffer 330 and the pulverized tissue sample 314 can be forced throughthe first separation element 306 and into extraction chamber 308 ofsample processing device 300. In some embodiments of the invention,following the filtration step, during the extraction step 366, thesolution of extraction buffer 330 and the pulverized tissue sample 314can be heated and then cooled to room temperature. In some embodiments,the filtration step 368 follows the extraction step 366. In someembodiments, during the filtration step 368, extracted nucleic acid 352can be filtered through the second separation element 310 and into thedilution chamber 312. In some embodiments of the invention, thepulverized tissue sample 314 can remain in the extraction chamber 308.In some embodiments, following the filtration step 368, during theneutralization step 370, a neutralization buffer 354 can be added todilution chamber 312 to neutralize isolated nucleic acid 352. In someembodiments, following the neutralization step 370, during a dilutionstep 372, water or a dilution buffer can be added to the dilutionchamber 312. In some embodiments, following these steps as describedabove, isolated nucleic acid 352 can be stored, and/or aspirated to aninline device to perform nucleic acid amplification, and/or or dispensedinto a sample vial that can be inserted into an amplification device.

FIG. 19 is a side view of sample processing device 400 in accordancewith some embodiments of the invention, and FIG. 20 is a cross-sectionalview of the sample processing device 400 along line A-A in FIG. 19. FIG.21A is a cross-sectional view of sample processing device 400A, oneembodiment of sample processing device 400 shown in FIGS. 1 and 2. FIG.21B is a cross-sectional view of sample processing device 400B, anotherembodiment of sample processing device 400 shown in FIGS. 1 and 2. Insome embodiments of the invention, the sample processing devices 400,400A, 400B are advantageous because they reduce the risk ofcontamination when pulverizing and denaturing a sample to prepare thesample for testing. Devices 400, 400A, 400B can eliminate the need toperform pulverization in one vessel and transfer the pulverized sampleinto another vessel for denaturing, thus reducing opportunities forcontamination of the sample.

In some embodiments of the invention, the sample processing devices 400,400A, and 400B can be disposable tubes. In some embodiments, the sampleprocessing devices 400, 400A, and 400B can include a cylindrical portion402 coupled or integrated with a tapered portion 404, cap 406, cap 408,ribs 410, and ring 412. In some embodiments of the invention, thecylindrical portion 402 and tapered portion 404 can be at leastpartially hollow. In some embodiments, the cap 406 can at leastpartially cover the end of cylindrical portion 402, and the cap 408 canat least partially cover the end of tapered portion 404. In someembodiments, the ribs 410 and ring 412 can be coupled to the cylindricalportion 402 of the sample processing device 400. Further, in someembodiments, the ribs 410 can extend along cylindrical portion 402. Someembodiments include a ring 412 that is located at the end of ribs 410proximate or adjacent to where the cylindrical portion 402 meets thetapered portion 404.

In some embodiments of the invention, the sample processing devices 400Aand 400B can be similar to sample processing device 400, except sampleprocessing device 400A can include a pulverizer 400A, and sampleprocessing device 400B can include a pulverizer 400B. As shown in FIG.21A, in some embodiments, the pulverizer 416A can be cylindricallyshaped. As shown in FIG. 21B, in some embodiments, the pulverizer 416Bcan be spherically shaped. The following description references sampleprocessing device 400A as shown in FIG. 21A, but the description canapply to both sample processing device 400A and sample processing device400B.

In some embodiments of the invention, the pulverizer 416A can becontained within cylindrical portion 402. In some embodiments, the ring412 can prevent the pulverizer 416A from sliding into the taperedportion 404 of the sample processing device 400A. In alternativeembodiments of the invention, the pulverizer 416A can be any shape thatcan be contained by ribs 410 and ring 412 within cylindrical portion 402of sample processing device 400A. In some embodiments, the ribs 412 canprevent the pulverizer 416A from touching the sides of sample processingdevice 400, and can allow the pulverizer 416A to slide withincylindrical portion 402. In some embodiments, the pulverizer 416A can beany non-reactive material such as glass, ceramic, stainless steel, orany non-reactive polymer.

In some embodiments of the invention, the sample processing device 400Acan enable a crude sample to be both pulverized and denatured in thesame vessel. In some embodiments, in order to pulverize and denature asample, the cap 406 can be removed from the end of cylindrical portion402, and pulverizer 416A can be placed within the cylindrical portion402. In some embodiments of the invention, the crude sample can beplaced into the cylindrical portion 402. The crude sample can be one ormore pieces of corn, one or more seed or seed portions, one or moreleaves or leave portions, combinations thereof, or any other solidbiological sample. In some embodiments of the invention, once the crudesample and pulverizer 416A are placed within cylindrical portion 402,the cap 406 can be placed back on the end of cylindrical portion 402. Inan alternative embodiment of the invention, the sample processing device400A can be manufactured with pulverizer 416A already contained withincylindrical portion 402, and the crude sample can be added into eitherthe cylindrical portion 402 through the cap 406 or the tapered portion404 through the cap 408.

In some embodiments of the invention, once the crude sample andpulverizer 416A are at least partially sealed within sample processingdevice 400A, the sample processing device 400A can be placed into aninstrument, such as a shaker, with the cap 406 facing down. In someembodiments, the instrument can shake the sample processing device 400Asuch that the pulverizer 416A can move against the ribs 410 and slidewithin cylindrical portion 402 to macerate the crude sample. In someembodiments, once the sample has been pulverized, the sample processingdevice 400A can be removed from the instrument. In some embodiments, thecap 408 can then be removed from the tapered portion 404, and adenaturing agent, such as sodium hydroxide, can be added into thetapered portion 404. In some embodiments, the tapered shape of thetapered portion 404 and ring 412 can prevent the pulverizer 416A fromsliding into the tapered portion 404 when the denaturing agent is added.In some embodiments, this can reduce the risk of contamination byensuring that pulverizer 416A is contained within cylindrical portion402, and is prevented from sliding out of the sample processing device400A. In some embodiments, after the denaturing agent has been added,the cap 408 can be placed back on the tapered portion 404.

In some embodiments, once the denaturing agent and pulverized sample areat least partially sealed within the sample processing device 400A, thesample processing device 400A can be placed back into the instrumentwith the cap 408 facing down. In some embodiments, the instrument canshake the sample processing device 400A such that the denaturing agentat least partially denatures the DNA within at least a portion of thepulverized sample. In some embodiments, the gaps between the ribs 410can allow the denaturing agent to flow within the cylindrical portion402 in addition to the tapered portion 404 in order to denature theentire pulverized sample. In some embodiments, once at least a portionof the DNA within at least a portion of the sample has been denatured,at least a portion of the denatured sample can be transferred into aseparate vessel for testing, or the portion of the denatured sample canbe tested in sample processing device 400A. In some embodiments, if thedenatured sample is transferred to a separate vessel, the cap 408 can beopened in order to transfer the sample. In some embodiments of theinvention, the tapered portion 404 and ring 412 can prevent thepulverizer 416A or pulverizer 416B from sliding out of sample processingdevice 400A when the denatured sample is being transferred.

FIG. 22 is a perspective view of sample processing device 500 inaccordance with some embodiments of the invention. In some embodiments,the sample processing device 500 can include tube 502 and cap 504. Insome embodiments of the invention, the tube 502 can include basket 506with grid 508 and tabs 510. In some embodiments, the tube 502 can alsoinclude a barcode 512, and can be filled with lysis buffer 514 that canlyse cells within a macerated sample prepared within the device 500 inorder to expose the DNA within the cells for further testing.

In some embodiments, the basket 506 can be a removable basket that sitsin tube 502 with tabs 510 hanging over the edge of the tube 502. In someembodiments, the cap 504 can include a punch edge 516 and fingers 518.In some embodiments, the sample processing device 500 can be used totake a sample of a crude biological material, such as a leaf, seed, orclip from an ear of corn, and force the sample through the grid 508 ofthe basket 506 to macerate the sample. In some embodiments, themacerated sample can be combined with the lysis buffer 514 to extract atleast some DNA from the sample for further testing.

In some embodiments, in order to take and process a sample with sampleprocessing device 500, some crude biological material can be placed ontop of tube 502, and the cap 504 can be coupled to the tube 502 to atleast partially seal the tube 502. In some embodiments, the cap 504 canbe a snap top cap. In some embodiments, as the cap 504 is snapped intoplace, the punch edge 516 can punch a sample out of the crude biologicalsample and into the basket 506. In some embodiments, the punch edge 516can be a ring-shaped sharp edge suitable for cutting a crude biologicalsample. In some embodiments, as the cap 504 is snapped into place, thefingers 518 can force the sample through the grid 508 of basket 506 tomacerate the sample. In some embodiments. In some embodiments, themacerated sample can then fall into lysis buffer 514. In someembodiments, the grid 508 and fingers 518 can be made of a polymer, ametal (e.g., such as aluminum or stainless steel), a composite material,a ceramic or glass, or combinations thereof.

In some embodiments of the invention, the closing cap 504 can cause thetabs 510 to break off of basket 506 such that basket 506 with anymacerated sample remaining on the grid 508 can fall into the lysisbuffer 514. In an alternative embodiment of the invention, the closingcap 504 can cause the tabs 510 to break off the basket 506 so that thecap 504 can be completely sealed on the tube 502, but the basket 506does not fall into the lysis buffer 514. In some embodiments of theinvention, this can enable the macerated sample to be kept separate fromlysis buffer 514 before combining the macerated sample and lysis buffer514. In some embodiments, the tabs 510 can ensure that the sampleprocessing device 500 can be used only once, thus avoiding potentialcombining of biological samples when collecting multiple samples intomultiple sample processing devices. In an alternative embodiment, sampleprocessing device 500 can be used without lysis buffer 514. In thisalternative embodiment, sample processing device 500 can be used topunch and macerate a sample and store the macerated sample for futuretesting.

FIG. 23 is a perspective view of sample processing device 520, anotherembodiment of sample processing device 500 in FIG. 22. In someembodiments, the sample processing device 520 can include a tube 522with internal threads 523 and cap 524 with external threads 525. In someembodiments, the cap 524 can include punch edge 536 and teeth 538. Insome embodiments, the tube 522 can include basket 526 with grid 528 andtabs 530. In some embodiments, the basket 526 can be a removable basketthat sits in tube 522 with tabs 530 hanging over the edge of tube 522.In some embodiments, the tube 522 can also include barcode 532, and canbe filled with lysis buffer 534.

In some embodiments of the invention, the sample processing device 520can function in substantially the same manner as sample processingdevice 500 in FIG. 22, except cap 524 can include teeth 538 instead offingers 518. Further, cap 524 can screw into the tube 522 instead ofsnapping onto tube 522; external threads 525 of cap 524 fit intointernal threads 523 of tube 522. In some embodiments, in order to takeand process a sample with sample processing device 520, crude biologicalmaterial can be placed on top of tube 522. As cap 524 is threaded intotube 522, punch edge 536 can punch a sample out of the crude biologicalsample into basket 526. Further, in some embodiments, as cap 524 isthreaded into tube 522, teeth 518 grind the sample against and/orthrough grid 528 of basket 526 to macerate the sample. In alternativeembodiments, teeth 518 can be another textured surface that can grind atleast a portion of the sample against and/or through grid 528 which canthen fall into the lysis buffer 534. In some embodiments, closing cap524 can cause the tabs 530 to break off of the basket 526 so that basket526 with any macerated sample remaining on grid 528 falls into lysisbuffer 534.

FIG. 24 is a perspective view of sample processing device 540, anotherembodiment of sample processing device 500 in FIG. 22. In someembodiments, the sample processing device 540 can include tube 542 andcap 544. In some embodiments, the tube 542 includes basket 546 with grid548 and tabs 550. In some embodiments, the basket 546 can be a removablebasket that sits in tube 542 with tabs 550 hanging over the edge of tube542. Further, in some embodiments, the tube 542 can also include barcode552, sodium hydroxide pouch 554, and permeable layer 555. Further, insome embodiments, the cap 544 can include a punch edge 556 and fingers558 with piercers 559.

In some embodiments of the invention, the sample processing device 540can function in substantially the same manner as sample processingdevice 500 in FIG. 22, except tube 542 includes sodium hydroxide pouch554 and permeable layer 555 instead of lysis buffer 514. Additionally,in some embodiments, fingers 558 include piercers 559. In someembodiments of the invention, in order to take and process a sample withsample processing device 540, crude biological material can be placed ontop of tube 542. In some embodiments, the cap 544 can be used to atleast partially seal the tube 542. In some embodiments, as the cap 544is snapped into place, the punch edge 556 can punch a sample out of thecrude biological sample into basket 546. In some embodiments, as the cap544 is snapped into place, the fingers 558 force the sample through grid548 of basket 546 to macerate the sample, and piercers 559 can puncturethe sodium hydroxide pouch 554. In the embodiment shown, cap 544includes four piercers on fingers 558. In alternative embodiments, cap544 can include a single piercer 559 or multiple piercers 559. In someembodiments of the invention, the piercers 559 can be sharp tips capableof puncturing the sodium hydroxide pouch 554.

In some embodiments of the invention, once the sodium hydroxide pouch554 is pierced, the sodium hydroxide and the macerated sample can passthrough the permeable layer 555 into the bottom of tube 542. In someembodiments of the invention, the permeable layer 555 can be a metal orplastic screen. In some embodiments, closing cap 544 can cause the tabs550 to break off of basket 546 with any macerated sample remaining ongrid 548 falls onto permeable layer 555. In some embodiments, thepermeable layer 555 can allow the sodium hydroxide and macerated sampleto pass through into the bottom of tube 542, but can prevent the basket546 from passing into the bottom of tube 542. In some embodiments, thesodium hydroxide can denatures DNA within the macerated sample in orderto expose the DNA within the cells for further testing.

FIG. 25 is a perspective view of sample processing device 560, anotherembodiment of sample processing device 500 in FIG. 22. In someembodiments, the sample processing device 560 can include tube 562 withinternal threads 563 and cap 564 with external threads 565. In someembodiments, the tube 562 can include basket 566 with grid 568 and tabs570. In some embodiments of the invention, the basket 566 can be aremovable basket that sits in the tube 562 with tabs 570 hanging overthe edge of tube 562. In some embodiments, the tube 562 also includesbarcode 572, sodium hydroxide pouch 574, and permeable layer 575.Further, in some embodiments, the cap 564 can include punch edge 576,teeth 578, and piercer 579.

In some embodiments of the invention, the sample processing device 560can function in substantially the same manner as sample processingdevice 540 in FIG. 24, except cap 564 includes teeth 578 instead offingers 558. Additionally, in some embodiments, the cap 564 can screwinto the tube 562 instead of snapping onto tube 562; external threads565 of cap 564 fit into internal threads 563 of tube 562. In someembodiments of the invention, in order to take and process a sample withsample processing device 560, crude biological material can be placed ontop of tube 562. In some embodiments, as cap 564 is threaded into thetube 562, the punch edge 576 can punch a sample out of the crudebiological sample into basket 566. In some embodiments, as cap 564 isthreaded into tube 562, the teeth 578 can grind the sample againstand/or through grid 568 of basket 566 to macerate the sample, andpiercer 579 can puncture the sodium hydroxide pouch 574. In someembodiments of the invention, the piercer 579 can be a needle-shapedprojection.

In some embodiments of the invention, once the sodium hydroxide pouch574 is pierced, sodium hydroxide and macerated sample can pass throughpermeable layer 575 into the bottom of tube 562. In some embodiments,closing the cap 564 can cause the tabs 570 to break off of basket 566with any macerated sample remaining on the grid 568 falling onto thepermeable layer 575. In some embodiments, the permeable layer 575 canallow the sodium hydroxide and macerated sample to pass through into thebottom of the tube 562 but can prevent the basket 566 from passing intothe bottom of tube 562.

FIG. 26 is a perspective view of sample processing device 580, anotherembodiment of sample processing device 500 in FIG. 22. Some embodimentsinclude a sample processing device 580 that includes tube 582 and cap584. In some embodiments, the tube 582 includes basket 586 with grid 588and tabs 590. In some embodiments, the basket 586 can be a removablebasket that sits in tube 582 with tabs 590 hanging over the edge of tube582. In some embodiments, the tube 582 can also include barcode 592. Insome embodiments, the cap 584 can include septum 593, sodium hydroxidepouch 594, permeable layer 595, punch edge 596, and fingers 558.

Some embodiments of the invention include a sample processing device 580that functions in substantially the same manner as sample processingdevice 540 in FIG. 24, except cap 584 can include sodium hydroxide pouch594 and septum 595, and the tube 582 does not include a sampleprocessing liquid. In some embodiments, in order to take and process asample with sample processing device 580, crude biological material canbe placed on top of the tube 582, and the cap 584 can be used to sealthe tube 582. In some embodiments, as cap 584 is snapped into place, thepunch edge 596 can punch a sample out of the crude biological sample andinto the basket 586. In some embodiments, as the cap 584 is snapped intoplace, the fingers 598 can force the sample through the grid 588 ofbasket 586 to macerate the sample. In some embodiments, closing the cap584 can cause the tabs 590 to break off of the basket 586 so that thebasket 586 with any macerated sample remaining on the grid 588 can fallinto the bottom of tube 582.

In some embodiments, after cap 584 has been snapped into place, apiercing device, such as a needle, can be inserted into the septum 593to pierce the sodium hydroxide pouch 594. In some embodiments of theinvention, the septum 593 can be a resealable septum. In someembodiments, once the sodium hydroxide pouch 554 is pierced, sodiumhydroxide passes through the permeable layer 595 and into the bottom oftube 582. In some embodiments, the permeable layer 595 can be a metal orplastic screen. In an alternative embodiment, the cap 584 can be made ofa compressible material such that sodium hydroxide pouch 594 can bepopped by applying pressure to cap 584.

FIG. 27 is a perspective view of sample processing device 600, anotherembodiment of sample processing device 500 in FIG. 22. In someembodiments, the sample processing device 600 can includes tube 602 withinternal threads 603 and cap 604 with external threads 605. In someembodiments, the tube 602 can includes basket 606 with grid 608 and tabs610. In some embodiments, the basket 606 can be a removable basket thatsits in tube 602 with tabs 610 hanging over the edge of tube 602. Insome embodiments, the tube 602 also includes barcode 612, and the cap604 includes septum 613, sodium hydroxide pouch 614, permeable layer615, punch edge 616, and teeth 618.

In some embodiments, the sample processing device 600 functions insubstantially the same manner as sample processing device 580 in FIG.26, except cap 604 includes teeth 618 instead of fingers 598.Additionally, in some embodiments, the cap 604 screws into the tube 602instead of snapping onto the tube 602; external threads 605 of cap 604fit into internal threads 603 of tube 602. In some embodiments, in orderto take and process a sample with sample processing device 600, crudebiological material is placed on top of tube 602. In some embodiments,as cap 604 is threaded into tube 602, punch edge 616 punches a sampleout of the crude biological sample into basket 606. In some embodiments,as cap 604 is threaded into tube 602, teeth 618 can grind the sampleagainst and/or through grid 618 of basket 606 to macerate the sample. Insome embodiments, closing cap 604 causes tabs 610 to break off of basket606 such that basket 606 with any macerated sample remaining on grid 608falls into the bottom of tube 602.

In some embodiments, after cap 604 has been closed into place, apiercing device, such as a needle, can be inserted into septum 613 topierce sodium hydroxide pouch 614. In some embodiments, once the sodiumhydroxide pouch 614 is pierced, sodium hydroxide passes throughpermeable layer 615 into the bottom of tube 602. In an alternativeembodiment, the cap 604 can be made of a compressible material where thesodium hydroxide pouch 614 can be popped by applying pressure to cap604.

It will be appreciated by those skilled in the art that while theinvention has been described above in connection with particularembodiments and examples, the invention is not necessarily so limited,and that numerous other embodiments, examples, uses, modifications anddepartures from the embodiments, examples and uses are intended to beencompassed by the claims attached hereto. Various features andadvantages of the invention are set forth in the following claims.

The invention claimed is:
 1. An inline sample processing devicecomprising: a first chamber configured to receive a tissue sample and anucleic acid extraction reagent, the first chamber configured to permitthe tissue sample to be pulverized therein; a second chamber in flowcommunication with the first chamber and configured to receive theextraction reagent and a portion of the pulverized tissue sample; afirst separation element disposed between the first chamber and thesecond chamber and configured to permit a portion of the pulverizedtissue sample and the extraction reagent to flow from the first chamberto the second chamber; a third chamber in flow communication with thesecond chamber, the third chamber having a plurality of ports configuredto allow a reagent to be delivered to the third chamber; and a secondseparation element disposed between the second chamber and the thirdchamber and configured to substantially prevent the pulverized tissuefrom entering the third chamber.
 2. A sample processing device forpulverization and denaturation, the sample processing device comprising:a hollow cylindrical portion; a hollow tapered portion; a first capconfigured and arranged to cover an end of the cylindrical portion; asecond cap configured and arranged to cover an end of the taperedportion; a plurality of ribs extending along the cylindrical portion; aring at an end of the plurality of ribs where the cylindrical portionmeets the tapered portion; and a pulverizer contained within thecylindrical portion between the ring and the first cap.
 3. The sampleprocessing device of claim 1, wherein the pulverizer is cylindrical orspherical.
 4. The sample processing device of claim 2, wherein thepulverizer includes at least one of glass, ceramic, stainless steel, anda polymer.
 5. The sample processing device of claim 2, wherein theplurality of ribs is configured and arranged to prevent the pulverizerfrom touching sides of the sample processing device and allows thepulverizer to slide within the cylindrical portion.
 6. The sampleprocessing device of claim 2, wherein the ring is configured andarranged to prevent the pulverizer from entering the tapered portion ofthe sample processing device.
 7. A method for pulverizing and denaturinga crude sample, the method comprising: placing a pulverizer in a sampleprocessing device, the sample processing device comprising: acylindrical portion; a tapered portion; a plurality of ribs extendingalong the cylindrical portion; and a ring at an end of the plurality ofribs where the cylindrical portion meets the tapered portion; placingthe crude sample in the cylindrical portion of the sample processingdevice; sealing the sample processing device; shaking the sampleprocessing device such that the pulverizer macerates the crude sample tocreate a pulverized sample; adding a denaturing agent into the taperedportion of the sample processing device; sealing the sample processingdevice; and shaking the sample processing device such that thedenaturing agent denatures DNA within the pulverized sample to create adenatured sample.
 8. The method of claim 7, and further comprisingtransferring the denatured sample into a testing vessel.
 9. The methodof claim 7, and further comprising testing the denatured sample in thesample processing device.